Show:
Codons and Anticodons
RNA
Translation I
tRNA
Translation II
100
What establishes the reading frame in RNA?
The AUG start codon
100
DAILY DOUBLE: Given that there are four different bases, what is the minimum number of nucleotides per amino acid required to specify all 20 amino acids?
3 (4^3 = 64 total possible codons, encompasses 20 AAs)
100
When IF-2 delivers the Met-tRNA to the P-site during translation initiation, does it require the use of a high energy bond? If yes, where does the high energy bond come from?
YES! It hydrolyzes GTP --> GDP + Pi
100
What class of enzymes load amino acids onto tRNAs?
aminoacyl tRNA synthetases
100
What is a "signal sequence" and what is its function?
Signal sequence is a short amino acid sequence on the polypeptide. It helps direct the protein to its appropriate location in the cell.
200
Describe how aminoacyl tRNAs interact with mRNA.
They pair with the mRNA through the use of their anticodon. The tRNA's anticodon is antiparallel to the mRNA's codon.
200
List and briefly describe two models for RNA editing given in the textbook.
1. The use of a special class of Guide RNAs that act as templates for the insertion of 4 U residues in the mRNA (see pg. 1112) 2. Cytosine spontaneous deamination to Uracil (or A to I)
200
During elongation, the amino acid in the A-site nucleophilically attacks the amino acid in the P-site. Name the enzyme that catalyzes this reaction. Is this reaction catalyzed by a protein enzyme or a ribozyme?
Peptidyl Transferase. It is a ribozyme.
200
Name 2 synthetic processes (cannot use activation of amino acids) you have studied in which pyrophosphate is generated. What is the significance of the fact that all cells possess pyrophosphatases that catalyze the hydrolysis reaction, PPi + H2O --> 2 Pi
DNA Replication and Transcription. The hydrolysis of Pyrophosphate drives equilibrium reactions (ex. activation of an amino acid) to favor the formation of more products. This is because you are hydrolyzing Pyrophosphate - removing product. Le Chatlier's Principle is key here.
200
All protein synthesis begins with the Met, AUG start codon. With this in mind, do all of the native proteins in your body begin at the N-terminus with Met?
No! Some proteins undergo post-translational modifications that remove Met, or part of the N-terminus. Therefore, not all of the mature protein products in your body begin with Met.
300
DAILY DOUBLE: If the genetic code were non-degenerate, only 20 of the triplet codons would encode amino acids. The remaining codons would then be stop codons. If the genetic code were non-degenerate, what fraction of mutations would create premature stop codons? Now, with the degenerate code, what fraction of mutations would create premature stop codons? (*There are 61 total non-stop codons)
41/61 (approx. 2/3) - non-degenerate code 3/61 - degenerate code
300
What is RNA World Hypothesis?
RNA world refers to the hypothesis that self-replicating RNA was the precursor to all life on earth today. Self-replicating RNA eventually gives rise to proteins, which begin to take over some of the replication responsibilities. Then, life evolved to use DNA over RNA because it is more stable than RNA for long-term information storage. (see pg. 1117 for more details).
300
What is the Shine-Dalgarno sequence? Explain its significance in prokaryotic translation initiation.
The Shine-Dalgarno sequence is on prokaryotic mRNA and base pairs with the 16S rRNA of the small subunit. It helps distinguish the start AUG codon from other Met codons because the start AUG is the one close to the Shine-Dalgarno sequence.
300
What is meant by the "second genetic code"?
The interaction between amino-acyl tRNA synthetases and tRNAs. Remember, amino-acyl tRNA synthetases must be specific not only for a specific amino acid but for certain tRNAs as well, adding an additional level of specificity to the process.
300
Do termination codons (UAA, UGA, UAG) encode for termination tRNAs? If no, what do they encode for?
They do NOT encode for termination tRNAs because there is no such thing. They encode for the release factor.
400
If the anticodon triplet of a tRNA recognized only one codon triplet through Watson-Crick base pairing at all three positions, cells would have a different tRNA for each amino acid codon. This however, is NOT the case. Could you discuss what is meant by the term "Wobble" and how it allows some tRNAs to recognize more than one codon?
"Wobble" refers to the first base of the anticodon (5'->3') and the third base of the codon. They pair loosely with one another, so this allows some tRNA to recognize more than one codon.
400
Distinguish between co- and post-transcriptional modification, and RNA editing.
RNA editing can involve addition, deletion, or alteration of nucleotides in the RNA in a way that affects the "meaning" of the transcript when it is translated. Co- and Post-transcriptional modification can include the 5' cap, poly-A tail, and splicing of introns. It is a process of converting Primary transcript into translatable, mature, mRNA.
400
Discuss the role of EF-Tu in proofreading.
EF-Tu is a elongation factor that is a GTPase. It helps bring the new amino-acyl tRNA into the A-site. It is important to know that hydrolyzing GTP --> GDP + Pi is actually kind of "slow". This is crucial for EF-Tu's proofreading ability. When the new tRNA is brought into the A-site, this time that it takes to try and hydrolyze GTP, allows for the checking of proper base pairing. If the tRNA does not properly base pair with the codon, it is removed. If it does, GTP is hydrolyzed and it stays. The key here is once GTP is hydrolyzed, you're done. That tRNA is not leaving.
400
DAILY DOUBLE: Using structures, describe in 2 steps how the activation of an amino acid for protein synthesis takes. Show the reactants, intermediates, and the products. *You don't need to know the structure of the nitrogenous base, just put a letter instead.
Jim, Dan, and Rakeeb can judge the structures here.
400
Does peptide bond formation require the input of energy?
YES. The required energy is released when the peptidyl-tRNA ester bond is broken.
500
The genetic code is robust against mutations at the 5' and the 3' end of the triplet nucleotide codon. If there is a mutation at the 3' end of the codon, what is the usual outcome? How will this likely affect the structure and function of the encoded protein?
The usual outcome is that you encode for the same amino acid (see pg. 1107). If the usual outcome occurs, this mutation will have no effect on structure and function of the encoded protein.
500
The genetic code is robust against mutations at the 5' and the 3' end of the triplet nucleotide codon. If there is a mutation at the 5' end of the codon, what is the usual outcome? How will this likely affect the structure and function of the encoded protein?
The usual outcome is that you will encode for an amino acid that has similar properties to the one you tried to encode for. Usually, this will have little to no effect on the structure and function of the encoded protein. However, if the intended amino acid is critical in the active site of the protein of interest, you could have a protein that does not properly function.
500
DAILY DOUBLE: Consider a bacterial gene for a hypothetical polypeptide comprised of 100 amino acid residues. Assuming that each mRNA molecule is translated 20-50 times, does the cell spend more energy in transcription or translation? *Go through the math.
More energy spent in translation. 600 high energy bonds used in transcription. 400 high energy bonds used in translation (x20-50 = way more high energy bonds used than in transcription)
500
Discuss how Ile-tRNA synthetase distinguishes between Ile and Val at high fidelity.
It deals with the two active sites on Ile-tRNA synthetase. Lets say Ile binds to Ile-tRNA synthetase, after it is activated to Ile-AMP, it is unable to bind to Ile-tRNA synthetase's second active site. If Val binds to Ile-tRNA synthetase, after it is activated to Val-AMP, it is actually small enough to fit into Ile-tRNA synthetase's second active site. Here, it is hydrolyzed to Val and AMP, and kicked off.
500
DAILY DOUBLE: Draw a structure of tRNA and label each of the "arms" and the H-bonded regions. Discuss the function of each.
Jim, Dan, and Rakeeb will go around.